Haplotype analysis on chromosome 6p of tumor necrosis factor alpha, vascular endothelial growth factor A, and interleukin-17F alleles associated with corneal transplant rejection.

OBJECTIVE: The aim of this work was to investigate single-nucleotide polymorphisms (SNPs) in multiple genes on chromosome 6p in corneal transplant recipients known to be at increased risk of failure through immunologic rejection (ie, "high-risk" corneal transplants). Tumor necrosis factor alpha (TNF-α) is a key immunoregulatory cytokine in the ocular environment, interacting with a variety of factors in a synergistic way and playing a crucial role in many stages of the inflammatory response. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors, supporting both hemangiogenesis and lymphangiogenesis, both key in transplant tolerance and rejection. Interleukin-17 (IL-17) is a multifunctional cytokine produced by T-helper 17 cells, exerting specific effector functions during an immune response. Association of SNPs in all 3 genes with corneal transplant outcome was therefore investigated. METHODS: Three hundred five corneal transplant recipients were followed for 3 years, and episodes of allograft rejection were recorded. With the use of patient DNA, 6 SNPs of 3 different genes on chromosome 6p were investigated. The TNF-α promoter SNP -308 G/A (rs1800629) was analyzed with the use of induced heteroduplex generation; 2 VEGF-A functional variants were analyzed, -2578 (rs699947) C/A and -1154 (rs1570360) G/A, with the use of Taqman genotyping assays; and 3 nonsynonymous IL-17F SNPs in exon 3 (negative strand), (rs2397084) A/G, (rs11465553) G/A, and (rs763780) A/G, were investigated with the use of direct sequencing. Haplotypes were inferred with the use of PHASE using positive strand alleles, and exact measures of association were determined with the use of Mid-P exact chi-square. RESULTS: Six common haplotypes were inferred, with the haplotype TNF-α (rs1800629), VEGF-A (rs699947), (rs1570360), IL-17F (rs763780), (rs11465553), and (rs2397084) ACGTCT having a significant association with corneal transplant rejection (odds ratio, 1.78; 95% confidence interval, 1.01-3.11; P = .04). CONCLUSIONS: The results suggest that patients carrying a combination of SNPs for TNF-α, VEGF-A, and IL-17F of ACGTCT haplotype may have an increased risk of corneal allograft rejection compared with patients carrying other haplotypes.

Functional tumor necrosis factor alpha polymorphisms and haplotype analysis in high-risk corneal transplantation.

BACKGROUND: Tumor necrosis factor alpha (TNF-α) plays a critical role in diverse cellular processes including ocular immune tolerance, inflammation, and allograft rejection. The ubiquitous transcription factor nuclear factor kappa B (NF-κB) regulates expression of numerous genes. Induction of the TNF-α pathway is involved in the inflammatory response and loss of transplant tolerance. OBJECTIVES: We investigated functional single nucleotide polymorphisms (SNPs) in the promoter region of TNF-α and an insertion/deletion (indel) polymorphism of NF-κB1 in corneal transplant recipients considered to be at increased risk of immunological rejection (ie, high-risk corneal transplantations) and looked for any associations with corneal transplantation outcome. PATIENTS AND METHODS: Three hundred eighty-four full thickness corneal transplant recipients were followed for 3 years and episodes of reversible and irreversible allograft rejection were recorded. Using DNA obtained from these patients, 5 SNPs located in the promoter region -1031 T/C rs1799964, -863 C/A (rs1800630), -857 C/T (rs1799724), -308 G/A (rs1800629), and -238 G/A (rs361525), and one SNP upstream from the transcription start site (+489) rs1800610 of TNF-α were analyzed using induced heteroduplex generation. A functional NF-κB1 indel (-94) was also investigated. Haplotypes were inferred by PHASE and associations with rejection were determined by chi-square analysis. RESULTS: The TNF-α haplotype TCTGGA was significantly associated with reduced risk of corneal graft rejection (Pc < .005) and TCTAGA was associated with increased risk of rejection (Pc < .005) in high-risk corneal transplants. There was no association with the NF-κB1 indel (Pc > .05). CONCLUSION: According to haplotype frequencies, our results suggest that the TCTGGA haplotype may confer additional protection against risk of immunological rejection whereas TCTAGA may increase risk of corneal allograft rejection in the high-risk setting. However, both haplotypes were relatively rare and thus would not warrant genotyping for individual patient selection for anti-TNF therapy.

Evaluation of NFKB1A variants in patients with knee osteoarthritis.

A key feature of osteoarthritis (OA) is articular cartilage loss mediated by numerous catabolic factors including pro-inflammatory cytokines. Cytokine expression is modulated by the nuclear factor κB (NF-κB) family of transcription factors that are in turn, regulated by the inhibitor of NF-κB IκBα encoded by NFKB1A. We examined eight, previously reported common germline polymorphisms to determine whether NFKB1A variants are associated with knee OA. Eight common single-nucleotide polymorphisms (SNPs) across the NFKB1A gene were genotyped in 189 cases with knee OA and 197 healthy controls. Allele, genotype and haplotype frequencies were compared between case and control groups and stratified according to gender due to the increased prevalence of female OA. Serum concentrations of four biochemical markers elevated in OA were compared with genotype for each knee OA case. None of the SNPs showed an association with knee OA; however, stratification of the data for gender showed an increased frequency of the rs8904 variant allele in the female knee OA case group (P = 0.02). Six common haplotypes were identified (H1-H6). H6 was marginally more prevalent in the knee OA group (P = 0.05). The rs8904 variant was associated with increased levels of hyaluronan (HA), a marker of synovial inflammation at 12 and 24 months compared to baseline levels. The nearby rs696 variant demonstrated increased levels of C-reactive protein (CRP) at 12 months and HA at 12 and 24 months. A reduction in CRP levels at 12 months was observed for the rs2233419 variant. These findings provide evidence for the association of NFKB1A variants and knee OA.

Association between CCL2 haplotypes and knee osteoarthritis.

We examined five single nucleotide polymorphisms (SNPs) and reconstructed 5-locus haplotypes of the CCL2 gene, in knee osteoarthritis (OA) cases and in controls. The CCL2 rs2857657 variant (G) allele was observed more frequently in female knee OA cases than in controls. One haplotype (H5) was observed exclusively in the control group (f = 2.3%). Genetic variation in the CCL2 gene may be associated with knee OA.

Tumour necrosis factor receptor superfamily member 11B polymorphisms and association with knee osteoarthritis in women.

We examined single-nucleotide polymorphisms (SNPs) across the tumour necrosis factor receptor superfamily member 11B (TNFRSF11B) gene and knee OA. We identified alleles in a VNTR region in intron 3 that was observed exclusively in women OA cases (P = 0.007, Pc = 0.042). Our results reveal that a previously unreported association between a VNTR genotype in TNFRSF11B and knee OA in women.

Evaluation of IL18 and IL18R1 polymorphisms: genetic susceptibility to knee osteoarthritis.

We examined single-nucleotide polymorphisms (SNPs) in IL18 and IL18/R1 genes and knee OA. IL18 rs1946518 wild-type allele was more frequently observed in cases (P = 0.04). Haplotype 1 was more frequently observed in cases (P = 0.04). Genetic variation in the promoter region of IL18, but not IL18R1, may be associated with OA.

Haplotype uncertainty in association studies.

Inferring haplotypes from genotype data is commonly undertaken in population genetic association studies. Within such studies the importance of accounting for uncertainty in the inference of haplotypes is well recognised. We investigate the effectiveness of correcting for uncertainty using simple methods based on the output provided by the PHASE haplotype inference methodology. In case-control analyses investigating non-Hodgkin lymphoma and haplotypes associated with immune regulation we find little effect of making adjustment for uncertainty in inferred haplotypes. Using simulation we introduce a higher degree of haplotype uncertainty than was present in our study data. The simulation represents two genetic loci, physically close on a chromosome, forming haplotypes. Considering a range of allele frequencies, degrees of linkage between the loci, and frequency of missing genotype data, we detail the characteristics of genetic regions which may be susceptible to the influence of haplotype uncertainty. Within our evaluation we find that bias is avoided by considering haplotype probabilities or using multiple imputation, provided that for each of these methods haplotypes are inferred separately for case and control populations; furthermore using multiple imputation provides the facility to incorporate haplotype uncertainty in the estimation of confidence intervals. We discuss the implications of our findings within the context of the complexity of haplotype inference for larger marker rich regions as would typically be encountered in genetic analyses.

Cytokine gene polymorphism in human disease: on-line databases, Supplement 3.

Within the past few years, the focus on cytokine single nucleotide polymorphism (SNP) function and association with human diseases has increased considerably. This third supplement to the Cytokine Gene Polymorphism in Human Disease: On-line database describes the positive associations of cytokine SNPs in human diseases described in articles published from 2002 up to 2005. A file containing a list of all SNPs investigated in this period of time and their association with human disease or expression pattern can be downloaded from the internet address http://www.nanea.dk/cytokinesnps/. The web pages also contain other features and downloads that could be useful when planning cytokine SNP association studies.

Haplotypes of the low-density lipoprotein receptor-related protein 5 (LRP5) gene: are they a risk factor in osteoarthritis?

OBJECTIVE: Several genome-wide scans have revealed an osteoarthritis (OA)-susceptibility locus on chromosome 11q in close proximity to the low-density lipoprotein receptor-related protein 5 (LRP5) gene. The regulation of bone mass is under the control of LRP5 and since increased bone mass is thought to play a role in the pathology of OA we examined LRP5 polymorphisms and haplotypes to determine if variants of this locus may predispose to OA. METHODS: A UK control population of 187 individuals was examined for five commonly occurring polymorphisms against a cohort of 158 DNAs from patients with knee OA. An additional UK cohort was also examined to confirm the findings of the first study; this second group consisted of 110 knee OA patients. Haplotype analysis was also performed on patient and control DNAs. RESULTS: A study of individual polymorphisms revealed no association with disease. However, haplotype analysis of the initial two populations revealed a common haplotype (C-G-C-C-A) that provided a 1.6-fold increased risk of OA (P(c)=0.021). The data obtained from the second cohort confirmed the initial findings, with a 1.6-fold increased risk observed within this cohort for the risk haplotype (P=0.012). CONCLUSIONS: A closer investigation of LRP5 and associated Wnt signalling molecules in OA will help determine disease aetiology and the development of novel treatment strategies that specifically target the bone compartment.

Vascular endothelial growth factor gene polymorphism and acute respiratory distress syndrome.

BACKGROUND: Non-cardiogenic pulmonary oedema is a characteristic feature of the acute respiratory distress syndrome (ARDS). The properties of vascular endothelial growth factor (VEGF) as a potent vascular permogen and mitogen have led to investigation of its potential role in this condition. Lower VEGF plasma levels have been linked to the presence of the T allele in the +936 CT polymorphism. We hypothesised that the presence of the T allele would be associated with the development and severity of ARDS. METHODS: A cohort of 137 normal subjects, 117 ventilated patients with ARDS, and 103 "at risk" of ARDS were genotyped for the VEGF+936 CT polymorphism. The severity of physiological disturbance and mortality was determined in the ventilated cohorts. RESULTS: The CT and TT genotype frequencies were increased in ARDS patients compared with both normal subjects (OR 2.01, 95% CI 1.13 to 3.58, p = 0.02) and those "at risk" (OR 2.05, 95% CI 1.02 to 2.20, p = 0.03). In patients with ARDS but not those "at risk", CT and TT genotypes were associated with a higher mean APACHE III score (80.9 (4.3) v 69.3 (2.9), p<0.05). CONCLUSION: These data support a role for VEGF in the pathogenesis of ARDS and its associated physiological derangement.

Extended haplotypes and linkage disequilibrium in the IL1R1-IL1A-IL1B-IL1RN gene cluster: association with knee osteoarthritis.

The interleukin-1 gene cluster is a key regulator in a number of chronic disease processes. We explored the linkage between nine polymorphic loci in the IL1R1 promoter, eight in the IL1A-IL1B-IL1RN gene complex, and their association with osteoarthritis (OA), a common complex disease associated with low-level inflammation. Using 195 healthy controls, we identified eight novel polymorphisms in the IL1R1 exon 1A region. We found limited LD between IL1R1 and the IL1A-IL1B-IL1RN cluster, although LD within these two individual groups was high. To test association with knee OA, we genotyped 141 patients from Bristol (UK) at the 17 loci. IL1R1 promoter haplotypes showed no association with disease. However, within the IL1A-IL1B-IL1RN complex, we identified a common haplotype conferring a four-fold risk of OA (P=0.00043; Pc=0.0043) and one IL1B-IL1RN haplotype conferring a four-fold reduced risk (P=0.0036; Pc=0.029). To replicate these associations, we subsequently examined 163 knee OA patients from London. Here, the effects of the haplotypes were confirmed: the risk IL1A-IL1B-IL1RN haplotype conferred a two-fold risk of OA (P=0.02), and the protective IL1B-IL1RN haplotype conferred a five-fold reduced risk of OA (P=0.0000008). These results may help to explain the genome-wide scan linkage data and functional observations concerning association between IL-1 and OA.

Discrimination of suballeles present at the TNFd microsatellite locus using induced heteroduplex analysis.

Polymorphism at the TNFd locus has been implicated in a number of disease association studies. The TNFd locus consists of three regions of (GA)(n) repeats separated by an imperfect repeat of two guanine bases. TNFd alleles are genotyped by the number of repeats in the first (GA)(n) repeat region, and until now the second repeat region had been thought to be nonpolymorphic. We report the existence of suballeles present within the TNFd microsatellite locus, detected using induced heteroduplex generator (IHG) technology. These alleles cannot be detected using conventional typing strategies as they represent altered distribution of the (GA)(n) repeats or sequence variation within the repeat. The suballeles affect the frequencies of the conventional d3 and d4 alleles leading to significantly altered allele frequencies. Some studies have associated the d3 and d4 alleles with disease outcome. We re-analysed one such study cohort using IHG technology and demonstrated a high proportion of incorrectly assigned TNFd3 alleles.

Determination of cytokine regulatory haplotypes by induced heteroduplex analysis of DNA.

Multiple single nucleotide polymorphisms (SNP) in the promoter region of the human interleukin-10 (IL-10) gene and in the signal/leader sequence of the human transforming growth factor beta 1 (TGF-beta1) gene, have been associated with susceptibility, severity and clinical outcome for a number of diseases. One common explanation for this, is that different haplotypes of these SNPs regulate the expression of the respective cytokines. Therefore, accurate determination of haplotypes by physical linkage analysis represents an important tool in investigating the pathogenesis of such diseases. Here, we demonstrate that the use of induced heteroduplex generators (IHGs) may be used to identify haplotypes within target sequences in the IL-10 and TGF-beta1 genes. Four haplotypes were observed within the IL-10 promoter region, consisting of -1082, -851, -819 and -592 SNPs. For the TGF-beta1 signal/leader sequence, we observed three haplotypes of the T869C (Leu10Pro) and G915C (Arg25Pro) SNPs. In both cases, all combinations of these haplotypes could be resolved unequivocally with a single IHG reagent.